Curcumin and curcuminoid inhibition of angiogenesis

ABSTRACT

Methods for treating diseases or disorders of the skin which are characterized by angiogenesis have been developed using curcumin and curcumin analogs. Based on the results obtained with curcumin, it has been determined that other angiogenesis inhibitors can also be used to treat these skin disorders. It has further been discovered that curcumin acts to inhibit angiogenesis in part by inhibition of basic fibroblast growth factor (bFGF), and thereby provides a means for treating other disorders characterized by elevated levels of bFGF, such as bladder cancer, using curcumin and other analogues which also inhibit bFGF. Representative skin disorders to be treated include the malignant diseases angiosarcoma, hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma and Karposi&#39;s sarcoma, and the non-malignant diseases or conditions including psoriasis, lymphangiogenesis, hemangioma of childhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis, tuberous sclerosis, pyogenic granulomas, recessive dystrophic epidermolysis bullosa, venous ulcers, acne, rosacea, eczema, molluscum contagious, seborrheic keratosis, and actinic keratosis.

The United States government has rights in this invention by virtue ofgrant R03 AR44947 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

The invention is generally in the field of methods of inhibitingangiogenesis, and more specifically is drawn to methods and compositionsfor inhibiting angiogenesis.

Current treatments of cancer and related diseases have limitedeffectiveness and numerous serious unintended effects. Based primarilyon chemical, radiation and surgical therapy, these treatments haveprogressed only incrementally during more than thirty years of intensiveresearch to discover the origins and devise improved therapies ofneoplastic diseases.

Current research strategies emphasize the search for effectivetherapeutic modes with less risk, including the use of natural productsand biological agents. This change in emphasis has been stimulated bythe fact that many of the consequences, to patients and their offspring,of conventional cancer treatment, including new cancers, mutations andcongenital defects, result from their actions on genetic material andmechanisms. Hong et al., J. Natl. Cancer Inst. Monogr. 17:49-53 (1995).Efforts continue to discover the origins of cancer at the genetic level,and correspondingly new treatments, but such interventions also may haveserious unanticipated effects.

The observation by Folkman that tumors are highly vascular, and theelucidation by him and others of a process termed angiogenesis throughwhich many tumors derive a blood supply by the generation ofmicrovessels, provided an important new avenue to therapy of cancer andother diseases and disorders. Folkman, Proc. Natl. Acad. Sci. U.S.A.95(16):9064-6 (1998); C. R. Acad. Sci. III 316(9):909-918 (1993).Angiogenesis has now been recognized in inflammatory lesions and benigntumors, in addition to malignant tumors.

Mammals are characterized by complex cardiovascular systems that enabletheir warm-blooded nature, internal embryonic and fetal development andsuccessful population of extreme habitats. The development of anextensive capillary system, specialized in each organ and tissue, is anessential feature of mammalian cardiovascular systems, to provideoptimal distribution of nutrients and other substances includinghormones and defensive agents. The metabolic and physiologic needs ofmammalian cells are met by their proximity to capillaries, and limitedresources may be diverted by imbalance of this supply system. Tortora,“Principles of Human Anatomy”, 5^(th) ed., pp. 371-372, Harper & Row,N.Y. (1989).

Angiogenesis results primarily from the development of new or lengthenedcapillaries, and larger microvessels. Capillaries are formed primarilyof specialized endothelial cells and the connective tissue layer towhich they adhere, the basement membrane. The proliferation ofendothelial cells and their migration and orientation to formcapillaries is recognized as the key process regulated in the control ofangiogenesis. Neovascularization is a form of angiogenesis marked byformation of blood vessels in a tissue or region previously devoid ofblood vessel supply, for example the cornea of the eye. The mechanismsinvolved in angiogenesis are quite complicated, however, and no oneappears to be the sole controlling mechanism.

Mammals have effective mechanisms to regulate this vital process.Stimulation of angiogenesis in adult mammals, other than as a part ofnormal tissue repair, pregnancy or the menstrual cycle, is abnormal andoften pathological. Many malignant tumors, benign tumors andinflammatory lesions have the ability to evade or mobilize theseregulatory mechanisms to support their growth and further malignantprogression.

Development of effective preventive and treatment means has beenhampered by inadequate understanding of the factors controlling thisprocess. The premise of therapeutic development for such conditions isthat effective treatment does not require destruction of the cells ortissues of origin. Reduction or prevention of the increased blood supplycan be sufficient to prevent their growth, and the manifestation of thecondition as a disease or pathological disorder.

This concept was initially rejected, but widespread recognition ofangiogenesis as a major factor in a variety of pathological conditionsand diseases, particularly cancer and pre-cancerous conditions, hasoccurred recently among scientists and businesses. It is estimated that184 million U.S. and European Union (EU) disease cases could benefitfrom treatment to inhibit angiogenesis that is inappropriate andpathological (anti-angiogenic therapy), in addition to an estimated 314million disease cases in the U.S. and EU that might benefit fromtreatment to stimulate angiogenesis, for example in cardiacrehabilitation. Thirty-one specific projects of pharmaceutical andbiotechnology companies to develop anti-angiogenic treatment werereported in Gen. Eng. News 18(17):1, 8, 34, 46 (1998).

It is an object of the present invention to provide methods of treatinga mammal having a disease or condition characterized by increasedangiogenesis.

It is a further object of the present invention to provide a method ofpreventing the initiation or progression of a disease or conditioncharacterized by increased angiogenesis in a mammal, especially skindiseases and diseases characterized by elevated basic fibroblast growthfactor.

SUMMARY OF THE INVENTION

Methods for treating diseases or disorders of the skin which arecharacterized by angiogenesis have been developed using curcumin andcurcumin analogs. Based on the results obtained with curcumin, it hasbeen determined that other angiogenesis inhibitors can also be used totreat these skin disorders. It was also discovered that curcumin acts toinhibit angiogenesis in part by inhibition of basic fibroblast growthfactor (bFGF), and thereby provides a means for treating other disorderscharacterized by elevated levels of bFGF, such as bladder cancer, usingcurcumin and other analogues which also inhibit bFGF.

Curcumin and demethoxycurcumin are the preferred agents for treatingthese disorders. The preferred means of administration is to apply thecurcumin topically, for example, as an ointment or hydrogel containingbetween one-half percent (0.5%) and five percent (5%) of the curcumin,or regionally, orally to treat disorders of the gastrointestinal tractor by instillation, to treat bladder or cervical cancer. In alternativeembodiments, the curcumin or its analogs can be implanted in the form ofone or more pellets of a pharmaceutically acceptable vehicleencapsulating or encorporating the curcumin, or by one or moreinjections of a pharmaceutically acceptable aqueous solution includingthe curcumin.

Representative skin disorders include the malignant diseasesangiosarcoma, hemangioendothelioma, basal cell carcinoma, squamous cellcarcinoma, malignant melanoma and Karposi's sarcoma, and thenon-malignant diseases or conditions including psoriasis,lymphangiogenesis, hemangioma of childhood, Sturge-Weber syndrome,verruca vulgaris, neurofibromatosis, tuberous sclerosis, pyogenicgranulomas, recessive dystrophic epidermolysis bullosa, venous ulcers,acne, rosacea, eczema, molluscum contagious, seborrheic keratosis, andactinic keratosis. Representative disorders characterized by increasedlevels of bFGF include bladder and cervical cancers.

As demonstrated in the examples, curcumin and its analogs are potentinhibitors of endothelial cell proliferation, a sensitive test of invitro antiangiogenic effectiveness, and also of cornealneovascularization; a sensitive and reliable test of in vivoantiangiogenic effectiveness. The examples demonstrate that thisinhibition is exerted directly on the endothelial cells that areprimarily involved in angiogenesis, and not indirectly through othereffects of these agents. The examples further demonstrate that curcuminand its analogs inhibit the stimulation of angiogenesis in vivo by basicfibroblast growth factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C describe the effect of curcumin on endothelial cellproliferation in the absence of basic fibroblast growth factor (bFGF;FIG. 1A), in the presence of bFGF (FIG. 1B) and in the absence of bFGF,where the endothelial cells have been transformed (FIG. 1C). The figuresare graphs of cell number versus concentration of curcumin (μM).

FIGS. 2A-2B describe the effect of curcumin on the extent ofbFGF-stimulated neovascularization in the mouse cornea (FIG. 2A), inrelation to bFGF-stimulated neovascularization in the absence ofcurcumin (FIG. 2B). The figures are graphs of vessel length (mm) andsector size (clock hours) comparing curcumin (10 μM) with control TPCPD,with both in the presence of 80 ng bFGF.

FIGS. 3A and 313 describe the effect of curcumin and other curcuminoids,tetrahydrocurcumin, bisdemethoxycurcumin, and demethoxycurcumin, oncorneal neovascularization, as measured by vessel length (FIG. 3A) andby sector size (FIG. 3B).

DETAILED DESCRIPTION OF THE INVENTION I. Disorders to be Treated

Disorders or diseases that can be treated with the angiogenesisinhibitors include those characterized by elevated levels of basicfibroblast growth factor (bFGF), and a number of dermatologicaldisorders.

Diseases and pathological disorders of the skin characterized byangiogenesis in humans include the malignant diseases angiosarcoma,hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma,malignant melanoma and Karposi's sarcoma, and the non-malignant diseasesor conditions psoriasis, lymphangiogenesis, hemangioma of childhood,Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis, tuberoussclerosis, pyogenic granulomas, recessive dystrophic epidermolysisbullosa, venous ulcers, acne, rosacea, eczema, molluscum contagious,seborrheic keratosis, and actinic keratosis.

Examples of disorders characterized by elevated levels of bFGF includebladder cancer (O'Brien, et al. Cancer Res. 57(1):136-140 (1997)) andcervical cancer (which is caused by a herpes papilloma virus, known toelicit elevated levels of bFGF).

II. Pharmaceutical Compositions

A. Angiogenesis Inhibitors

Several different classes of compounds have been determined to be usefulas inhibitors of angiogenesis. These include collagenase inhibitors suchas metalloproteinases and tetracyclines such as minocycline, naturallyoccurring peptides such as endostatin and angiostatin, described forexample in U.S. Pat. No. 5,733,876 to O'Reilly, et al., U.S. Pat. No.5,290,807, and U.S. Pat. No. 5,639,725, fungal and bacterialderivatives, such as fumagillin derivatives like TNP-470, the sulfatedpolysaccharides described in U.S. Pat. No. 4,900,815 to Tanaka, et al.and the protein-polysaccharides of U.S. Pat. No. 4,975,422 to Kanoh, etal. and synthetic compounds such as the 2,5-diaryltetrahydrofurans ofU.S. Pat. No. 5,629,340 to Kuwano, et al., aminophenylphosphonic acidcompounds of U.S. Pat. No. 5,670,493 to Cordi, et al., the 3-substitutedoxindole derivatives of U.S. Pat. No. 5,576,330 to Buzzetti, et al., andthalidomides of U.S. Pat. No. 5,712,291 to D'Amato.

The antibiotics that are useful as angiogenesis inhibitors are thosehaving collagenase inhibitory activity. These include the tetracyclinesand chemically modified tetracyclines (CMTs), and three ringedtetracycline homologs, that have the ability to inhibit collagenase butdiminished antibacterial activity. Examples of commercially availabletetracyclines include chlotetracyline, demeclyeycline, doxycycline,lymecycline, methacycline, minocycline, oxytetracycline,rolitetracycline, and tetracycline. The active salts, which are formedthrough protonation of the dimethylamino group on carbon atom 4, existas crystalline compounds. These are stabilized in aqueous solution byaddition of acid.

Minocycline, a semisynthetic tetracycline antimicrobial, described byMartell, M. J., and Boothe, J. H. in J. Med. Chem., 10: 44-46 (1967),and Zbinovsky, Y., and Chrikian, G. P. Minocycline. In: K. Florey (ed.),Analytical Profiles of Drug Substances, pp. 323-339 (Academic Press, NY1977), the teachings of which are incorporated herein, hasanticollagenase properties, as reported by Golub. L. M., et al., J.Periodontal Res., 18: 516-526 (1983); Golub, L. M., et al., J.Periodontal Res. 19: 651-655 (1984); Golub, L. M., et al., J.Periodontal Res. 20: 12-23 (1985); and Golub, L. M., et al., J. Dent.Res., 66: 1310-1314 (1987). Minocycline, first described in 1967, isderived from the naturally produced parent compounds chlortetracyclineand oxytetracycline. The chemically modified tetracyclines are describedby U.S. Pat. No. 4,704,383 to McNamara, et al., U.S. Pat. No. 4,925,833to McNamara, et al., and U.S. Pat. No. 4,935,411 to McNamara, et al.,the teachings of which are incorporated herein.

Other exemplary anti-angiogenic compounds include penicillamine and somecytokines such as IL12.

Angiogenesis inhibitors may be divided into at least two classes. Thefirst class, direct angiogenesis inhibitors, includes those agents whichare relatively specific for endothelial cells and have little effect ontumor cells. Examples of these include soluble vascular endothelialgrowth factor (VEGF) receptor antagonists and angiostatin. Basicfibroblast growth factor (bFGF) is a potent, direct angiogenic factor,which has been shown to be a strong stimulus for both endothelialproliferation and migration, in vitro and in vivo. The activity of bFGFoh endothelial cells may be due in part to stimulation of protein kinaseC. Shing et al., Science 223:1296-1299 (1984); Kent et al., Circ. Res.77:231-238 (1995). Blockage of bFGF's stimulation of endothelial cellscan inhibit angiogenesis.

Indirect inhibitors may not have direct effects on endothelial cells butmay down-regulate the production of an angiogenesis stimulator, such asVEGF. Arbiser et al., Molec. Med. 4:376-383 (1998). VEGF has been shownto be up-regulated during chemically induced skin carcinogenesis; thisis likely due to activation of oncogenes, such as H-ras. Arbiser et al.,Proc. Natl. Acad. Sci. U.S.A. 94:861-866 (1997); Larcher et al., CancerRes. 56:5391-5396 (1996); Kohl et al., Nature Med. 1:792-797 (1995).Examples of indirect inhibitors of angiogenesis include inhibitors ofras-mediated signal transduction, such as farnesyltransferaseinhibitors.

Direct inhibition of endothelial cell proliferation can be assayed incell culture systems, in which the effects of specific factors whichcontrol the complex process of angiogenesis can be studied. Effectsdiscovered in such in vitro systems can then be studied in in vivosystems. Kenyon et al., Invest. Opthalmol. 37:1625-1632 (1996).

Curcumin (diferuloylmethane) and certain of its analogs, together termedcurcuminoids, are well known natural products, recognized as safe foringestion by and administration to mammals including humans. Bille etal., Food Chem. Toxicol. 23:967-971 (1985). Curcumin is a yellow pigmentfound in the rhizome of Curcuma longa, the source of the spice turmeric.Turmeric has been a major component of the diet of the Indiansubcontinent for several hundred years, and the average dailyconsumption of curcumin has been found to range up to 0.6 grams for someindividuals, without reported adverse effects. Food-grade curcuminconsists of the three curcuminoids in the relative amounts: 77%curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin.Thimmayamma et al., Indian J. Nutr Diet 20:153-162 (1983); Bille et al.,Food Chem. Toxicol. 23:967-971 (1985). The fully saturated derivativetetrahydrocurcumin is also included in the term curcuminoid.

Curcumin can be obtained from many sources, including for exampleSigma-Aldrich, Inc. The curcumin analogs demethoxycurcumin,bisdemethoxycurcumin and tetrahydrocurcumin can also be obtained frommany sources, or readily prepared from curcumin by those skilled in theart.

Curcumin has been used in indigenous Indian medicine for several hundredyears, as a topical agent for sprains and inflammatory conditions, inaddition to oral use to promote health and treat digestive and otherdisorders. Absorption of ingested or orally administered curcumin isknown to be limited, and absorbed curcumin is rapidly metabolized.Govindarajan, CRC Critical Rev. Food Sci Nutr. 12:199-301 (1980); Rao etal., Indian J. Med. Res. 75:574-578 (1982).

Numerous effects of the ingestion or oral administration of thecurcuminoids have been reported, based on controlled research,population studies, case reports and anecdotal information. Evidence ofchemopreventive activity of curcumin administered orally has led toclinical trials sponsored by the National Cancer Institute, regardingprevention of cancer. Kelloff et al., J. Cell. Biochem. Suppl. 26:1-28(1996). Oral administration of curcumin to mice treated with skin andcolon chemical carcinogens has been shown to result in a decreasedincidence and size of induced tumors compared with control mice. Huang,et al., Cancer Res. 54:5841-5847 (1994); Huang et al., Carcinogenesis16:2493-2497 (1995); Huang et al., Cancer Lett. 64:117-121; Rao et al.,Cancer Res. 55:259-266 (1995); Conney et al., Adv Enzyme Regul. 31:385-396 (1991).

Huang, et al. found that the oral administration of three curcuminoidcompounds curcumin, demethoxycurcumin and bisdemethoxycurcumin were ableto inhibit phorbol ester-stimulated induction of ornithine decarboxylaseand promotion of mouse skin initiated with 7,12-dimethylbenzanthracene(DMBA). These compounds also inhibited phorbol ester-mediatedtransformation of JB6 cells. The saturated derivative tetrahydrocurcuminwas less active than the unsaturated analogs in these assays. Huang etal., Carcinogenesis 16:2493-2497 (1995).

The mechanism or mechanisms of curcumin's chemopreventive activitieswere not previously understood, although it was recognized as anantioxidant and was known to exhibit antimutagenic activity in the AmesSalmonella test and to produce biochemical effects similar to those ofthe polyphenols, chemopreventive agents found in green tea. Stoner, J.Cell. Biochem. Suppl. 22:169-180 (1995). Curcumin has been demonstratedto inhibit several signal transduction pathways, including thoseinvolving protein kinase, the transcription factor NF-kB, phospholipaseA2 bioactivity, arachidonic acid metabolism, antioxidant activity, andepidermal growth factor (EGF) receptor autophosphorylation. Lu et al.,Carcinogenesis 15:2363-2370 (1994); Singh et al., J. Biol. Chem.270:24995-25000 (1995); Huang et al., Proc. Natl. Acad. Sci. U.S.A.88:5292-5296 (1991); Korutla et al., Carcinogenesis 16:1741-1745 (1995);Rao et al., Carcinogenesis 14:2219-2225 (1993).

Because of the complexity of the factors that regulate or effectangiogenesis, and their specific variation between tissues and accordingto circumstances, the response to a specific agent may be different oropposite, in different tissues, under different physiological orpathological conditions and between in vitro and in vivo conditions. Forexample, U.S. Pat. No. 5,401,504 to Das et al., discloses that oral ortopical administration of turmeric to animals including humans promoteswound healing, and postulates that it acts in part through stimulationof angiogenesis, although this postulate was not experimentallyverified. Administration of curcumin has been reported to inhibit smoothmuscle cell proliferation in vitro. Huang, et al., European J. Pharmac.221:381-384 (1992). U.S. Pat. No. 5,891,924 to Aggarwal discloses thatoral administration of curcumin to animals inhibits activation of thetranscription factor NF-kB, and claims its use in pathophysiologicalstates, particularly specific conditions involving the immune system.Several biochemical actions of curcumin were studied in detail, but nosingle action was reported to be responsible for these effects ofcurcumin. Singh et al. reported that curcumin inhibits in vitroproliferation of human umbilical vein endothelial cells (HUVEC) andsuggested that it might have anti-angiogenic activity. However, thisinhibition was independent of basic fibroblast growth factor stimulationof the proliferation of endothelial cells, and in vivo studies were notreported. Singh et al., Cancer Lett. 107:109-115 (1996). Thaloor et al.disclosed inhibition by curcumin of HUVEC growth and formation of tubestructures on Matrigel, in a model of capillary formation, and ascribedthis inhibition to modulation of metalloproteinases of the HUVEC.Thaloor et al., Cell Growth Differ. 9:305-312 (1998).

As demonstrated by the examples, these are not the mechanism involved ininhibition of angiogenesis as described herein, and accordingly, thedisorder to be treated and the dosage and means of administration aredifferent, based on the role of curcuminoids in inhibiting bFGF.

B. Carriers

Pharmaceutical compositions containing the angiogenesis inhibitor areprepared based on the specific application. Application can be eithertopical, localized, or systemic. Any of these compositions may alsoinclude preservatives, antioxidants, antibiotics, immunosuppressants,and other biologically or pharmaceutically effective agents which do notexert a detrimental effect on the normal tissue to be treated.

Compositions for local or systemic administration will generally includean inert diluent. Solutions or suspensions used for parenteral,intradermal, subcutaneous, or topical application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The parentalpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Systemic Carriers

Inhibitors can be systemically administered either parenterally orenterally. The composition can be administered by means of an infusionpump, for example, of the type used for delivering insulin orchemotherapy to specific organs or tumors, by injection, or by depousing a controlled or sustained release formulation. In a preferredsystemic embodiment, drugs are administered orally, in an entericcarrier if necessary to protect the drug during passage through thestomach.

The angiogenic inhibitors can be administered systemically by injectionin a carrier such as saline or phosphate buffered saline (PBS) ororally, in the case of an inhibitor such as thalidomide, in tablet orcapsule form. The tablets or capsules can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; or a glidant such as colloidal silicon dioxide. When thedosage unit form is a capsule, it can contain, in addition to materialof the above type, a liquid carrier such as a fatty oil. In addition,dosage unit forms can contain various other materials which modify thephysical form of the dosage unit, for example, coatings of sugar,shellac, or other enteric agents.

Local or Topical Carriers

The angiogenic inhibitors can also be applied locally or topically, in acarrier such as saline or PBS, in an ointment or gel, in a transdermalpatch or bandage, or controlled or sustained release formulation. Localadministration can be by injection at the site of the injury, or byspraying topically onto the injury. The inhibitor can be absorbed into abandage for direct application to the wound, or released from sutures orstaples at the site. Incorporation of compounds into controlled orsustained release formulations is well known.

For topical application, the angiogenesis inhibitor is combined with acarrier so that an effective dosage is delivered, based on the desiredactivity, at the site of application. The topical composition can beapplied to the skin for treatment of diseases such as psoriasis. Thecarrier may be in the form of an ointment, cream, gel, paste, foam,aerosol, suppository, pad or gelled stick. A topical composition for useof an ointment or gel consists of an effective amount of angiogenesisinhibitor in an ophthalmically acceptable excipient such as bufferedsaline, mineral oil, vegetable oils such as corn or arachis oil,petroleum jelly, Miglyol 182, alcohol solutions, or liposomes orliposome-like products.

In a preferred form for controlled release, the composition isadministered in combination with a biocompatible polymeric implant whichreleases the angiogenesis inhibitor over a controlled period of time ata selected site. Examples of preferred biodegradable polymeric materialsinclude polyanhydrides, polyorthoesters, polyglycolic acid, polylacticacid, polyethylene vinyl acetate, and copolymers and blends thereof.Examples of preferred non-biodegradable polymeric materials includeethylene vinyl acetate copolymers. These can be prepared using standardtechniques as microspheres, microcapsules, tablets, disks, sheets, andfibers.

An implantable pellet is the preferred mode of local administration ofthese agents to tissues. The preferred concentration of curcuminoidagent delivered locally to the target tissue is greater than 10micromolar, preferably 10-50 micromolar.

III. Methods for Treatment

For the treatment of skin disorders, the angiogenesis inhibitors areadministered topically or regionally. In a preferred embodiment, theinhibitors are administered in an ointment, salve or otherpharmaceutically acceptable carrier. For treatment of certain disorderscharacterized by elevated levels of bFGF, the angiogenesis inhibitors,preferably curcumin or demethoxycurcumin or another curcuminoidcompound, or a combination of two or more curcuminoid compounds, isapplied topically in diseases or pathologic conditions of the skin, orlocally in other tissues, to treat cancer, pre-malignant conditions andother diseases and conditions in which angiogenesis occurs. Thepreferred means of administration is to apply the curcumin topically,for example, as an ointment or hydrogel containing between one-halfpercent (0.5%) and five percent (5%) of the curcumin, or regionally,orally to treat disorders of the gastrointestinal tract or byinstillation, to treat bladder or cervical cancer.

The administration of these agents topically or locally may also used toprevent initiation or progression of such diseases and conditions. Forexample, a curcuminoid formulation may be administered topically or byinstillation into a bladder if a biopsy indicated a pre-cancerouscondition or into the cervix if a Pap smear was abnormal or suspicious.

The angiogenesis inhibiting formulation is administered as required toalleviate the symptoms of the disorder. Assays can be performed todetermine an so effective amount of the agent, either in vitro and invivo. Representative assays are described in the examples providedbelow. Other methods are known to those skilled in the art, and can beused to determine an effective dose of these and other agents for thetreatment and prevention of diseases or other disorders as describedherein.

The present invention will be further understood by reference to thefollowing non-limiting examples.

As demonstrated in the examples, curcumin inhibits basic fibroblastgrowth factor (bFGF)-induced proliferation of endothelial cells in vitroand angiogenesis in vivo. The effect of curcumin and curcumin analogswith known differential chemopreventive activities, demethoxycurcumin,tetrahydrocurcumin, and bisdemethoxycurcumin, on in vivo angiogenesiswas also demonstrated. Curcumin had a strong antiproliferative effect onendothelial cells, with a steep curve occurring between 5 and 10 μM.This was true both in the presence or absence of bFGF, and thisinhibition could not be overcome by the immortalizing ability of SV40large T antigen. The corneal neovascularization assay, which measuresincreased vessel length and density in vivo, in response to a bFGFpellet placed in the normally avascular cornea, has proven useful in theconfirmation and characterization of multiple angiogenesis inhibitors.The inhibition of bFGF-mediated corneal neovascularization by curcuminand its derivatives is evidence that curcumin is a direct angiogeniesisinhibitor in vivo. This inhibition was not due to dilution of bFGF, asadministration of a structurally related inactive compound,tetraphenylcyclopentadienone (TPCPD), had no effect on bFGF-inducedcorneal neovascularization. The lack of inhibition of TPA-mediated VEGFproduction further supports the role of curcumin as a directangiogenesis inhibitor.

The following materials and methods were used in the examples.

Materials and Methods

Endothelial Proliferation Assays

Bovine capillary endothelial cells were isolated according to the methodof Folkman, et al., Proc. Nat. Acad. Sci. U.S.A. 76:5217-5221 (1979),and were plated at a concentration of 10,000 cells/well in gelatinized24-well dishes. The primary endothelial cells were cultured inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovineserum and grown at 37° C. in 10% CO2. Twenty-four hours after plating,cells were treated with curcumin in the presence or absence of bFGF.After 72 hours of treatment, cells were counted using a Coulter counter.Cell counts for each condition were repeated in triplicate and in thepresence or absence of 1 ng/ml bFGF.

Similarly, MSI (ATCC CCRL 2279) endothelial cells, which are a SV40large T antigen immortalized murine endothelial cell line, were alsoplated at a concentration of 10,000 cells/well in nongelatinized 24-welldishes. MSI cells do not require endothelial mitogens for growth andwere cultured in DMEM supplemented with 5% fetal calf serum (FCS). Cellswere counted after a 72-hour exposure to curcumin with the same methodused for the bovine capillary endothelial cells.

Corneal Neovascularization

C57BL6 male mice (5-6 weeks old) were anesthetized with methoxyfluraneprior to implantation of pellets and with 0.5% proparacaine. A central,intrastromal linear keratotomy was performed with a surgical blade, anda lamellar micropocket was prepared according to the method of Kenyon,et al. (1996). The pellet was advanced to the end of the pocket.Erythromycin ointment was placed on the operated eye to preventinfection. Eyes were examined by slit lamp on days 3-6 afterimplantation under general anesthesia. Corneal angiogenesis was assayedthrough two measurements.

Vessel length is the length of the vessel from the corneal limbus as itgrows toward the pellet containing bFGF.

Sector size is a measurement of neovascularized area of the cornea. Thecornea is viewed as a circle that can be divided into twelve sectors of30 degrees span each, analogous to the division of a clock face intotwelve hours. Thus, neovascularization of a sector corresponding to onefourth of the cornea would be recorded as a sector size measurement ofthree. This system of measurement, recording sector sizes as “clockhours”, was established by Kenyon et al., Invest. Opthalmol.37:1625-1632 (1996).

Production of VEGF mRNA in HaCaT Keratinocytes

HaCaT keratinocytes were grown in (DMEM) (JRH) supplemented with 5% FCS(Hyclone, Logan, Utah) in 25 cm² flasks. One hour prior to stimulationwith 12-O-tetradecanoylphorbol-13-acetate (TPA), cells were switched toserumless media supplemented with 10 μM curcumin or an equal quantity ofethanol (final concentration 0.1%). TPA was added to a finalconcentration of 5 ng/ml and cells were incubated for three hours at 37°C. and harvested, and their RNA was extracted with guanidiniumthiocyanate/phenol

Phase II Enzyme Induction

The ability of curcumin derivatives to induce phase II activities wasmeasured by assaying quinone reductase [NAD(P)H:(quinone-acceptor)oxidoreductase, EC1.6.99.2] in murine Hepac1c7 cells. Serial dilutionsof curcumin, curcumin derivatives, and tetraphenylcyclopentadienone(TPCPD) were added, and the concentration of compound required to doublethe specific activity (CD) was calculated according to the method ofProchaska, et al., Proc. Natl. Acad. Sci. U.S.A. 89:2394-2398 (1992).

Materials

Curcumin, TPA and TPCPD were obtained from Sigma-Aldrich, Inc. Curcuminanalogs (bisdemethoxycurcumin, demethoxycurcumin and tetrahydrocurcumin)were provided by Dr. A. R. Conney of Rutgers—The State University of NewJersey.

C57BL6 mice were obtained from Charles River Laboratories. The MSItransformed cells were developed by Dr. J. L. Arbiser and deposited withthe ATCC (ATCC CCRI 2279).

Implant Pellets

Pellets were prepared according to a modification of the method ofKenyon, et al. Invest. Opthalmol. Vis. Sci. 37:1625-1632 (1996). Anaqueous solution of 18 mcg of basic fibroblast growth factor (SciosNova, Mountain View, Calif.) was evaporated to dryness under reducedpressure in the presence of 10 mg of sucralfate (Bukh Meditec, Vaerlose,Denmark) Ten microliters of 12% hydron and 10 mg of curcumin or curcuminanalog were then added, and the homogenous mixture was deposited onto asterile 15×15 mm 3-300/50 Nylon mesh (Tetko, Lancaster, N.Y.) and airdried. Once the mixture was dry, the mesh was manually dissociated toyield 225 pellets. Each pellet contained 80 ng of bFGF and 44 μg ofcurcumin or curcumin analog. Pellets containing hydron in the absence ofbFGF do not cause neovascularization, so pellets prepared in the absenceof bFGF were not used in this study. The approximate pore size was0.4×0.4 mm. Both sides of the mesh were covered with a thin layer ofhydron.

Isotopically Labelled Antisense Riboprobe

A plasmid containing the coding region of human vascular endothelialgrowth factor (VEGF) 121 was obtained from H. Welch (University ofFreiburg, Germany) and used to generate P³²-labeled antisense riboprobeas per manufacturers protocols (Ambion, Austin, Tex.). RNAse protectionassays were performed according to the method of Hod, Biotechniques13:852-853 (1992). Protected fragments were separated on gels of 5%acrylamide, 8 M urea, 1× Tris-borate buffer, and quantified with aphosphorimager (Molecular Dynamics, Sunnyvale, Calif.). An 18 Sriboprobe was included in each sample to normalize for variations inloading and recovery of RNA.

Measurement and Analysis

Significant differences between two groups were determined using anunpaired, two-tailed Student's t-test. Results are expressed as the meanplus or minus the standard error of the mean.

EXAMPLE 1 Curcumin Inhibition of Endothelial Cell Proliferation isDependent on Curcumin Dose and the Presence or Absence of BasicFibroblast Growth

Endothelial cells were stimulated to proliferate in the presence of 1ng/ml bFGF. Curcumin was added in concentrations ranging from 0.5 to 10μM to primary endothelial cells.

FIGS. 1A-C describe the effect of curcumin on endothelial cellproliferation in the absence of basic fibroblast growth factor (FGF;FIG. 1A), in the presence of bFGF (FIG. 1B) and in the absence of bFGF,where the endothelial cells have been transformed (FIG. 1C). A steepdecrease in cell number was seen at 10 μM. No evidence of cytotoxicitywas observed, and the number of cells at the end of treatment was notsignificantly less than the number cells originally plated. Thisdecrease in proliferation due to curcumin concentration of 10 μM wasobserved in both the presence or absence of bFGF.

In addition, curcumin was able to inhibit the growth of endothelialcells immortalized by SV40 large T antigen, with a similar dose responseas seen with primary endothelial cells.

EXAMPLE 2 Curcumin Inhibition of Corneal Neovascularization is Dependenton the Presence of Basic Fibroblast Growth Factor

The ability of curcumin to inhibit bFGF-induced cornealneovascularization in vivo was measured. Pellets were preparedcontaining 80 ng of bFGF and curcumin, or a control aromatic ketone,tetraphenylcyclopentadienone (TPCPD). TPCPD was added to rule out thepossibility that the inhibition of neovascularization due to curcuminwas not secondary to dilution. Neovascularization was assessed by slitlamp at 5 days after implantation, and the corneas were photographed.

FIGS. 2A-2B describe the effect of curcumin on the extent ofbFGF-stimulated neovascularization in the mouse cornea (FIG. 2A), inrelation to bFGF-stimulated neovascularization in the absence ofcurcumin (FIG. 2B). There was no difference in neovascularization inmice containing bFGF pellets in the presence or absence of TPCPD. Boththe vessel length and sectpr sizes were significantly reduced in thepresence of curcumin.

EXAMPLE 3 Curcumin and Other Curcumin Analog Inhibition of CornealNeovascularization in the Presence of Basic Fibroblast Growth Factor isDependent on the Dose and Structure of the Curcuminoid

Three curcumin analogs were assayed for their ability to inhibitbFGF-induced corneal neovascularization as described above.

FIGS. 3A and 3B describe the effect of curcumin and other curcuminoids,tetrahydrocurcumin, bisdemethoxycurcumin, and demethoxycurcumin, oncorneal neovascularization, as measured by vessel length (FIG. 3A) andby sector size (FIG. 3B). All analogs showed inhibitory activity, withdemethoxycurcumin showing the greatest activity on both sector size andvessel length, tetrahydrocurcumin having the least effect on sectorsize, and bisdemethoxycurcumin having the least effect on vessel length.All of the curcumin analogs showed significant inhibition ofbFGF-mediated neovascularization compared with control pellets.

EXAMPLE 4 Curcumin does not Inhibit Vascular Endothelial Growth FactormRNA Production in Transformed Keratinocytes

HaCaT cells are derived from spontaneously transformed humankeratinocytes. In order to determine whether curcumin could inhibitproduction of angiogenesis factors by relevant tumor cells as well asdirectly inhibit endothelial function; HaCaT cells were treated withtetradecanoylphorbol ester (TPA) in the presence or absence of curcuminand expression of VEGF mRNA was measured.

TPA caused a 2.5-fold increase in VEGF mRNA. This increase was notinhibited by curcumin. Thus the primary antiangiogenic effect ofcurcumin is directly on endothelium, rather than inhibition ofproduction of VEGF, an important angiogenic factor.

EXAMPLE 5 Inhibition of Corneal Neovascularization by Curcumin and OtherCurcuminoids does not Correlate with the Induction of Phase II Enzymesby Curcumin and Other Curcuminoids

Several plant-derived compounds with anticancer and chemopreventiveactivities also show the ability to induce phase II detoxifying enzymes,including quinone reductase. To determine whether the antiangiogenicactivities of curcumin derivatives correlated with the ability to inducequinone reductase activity, the concentration needed to double thespecific activity value (CD) was determined.

All of the curcumin analogs studied except tetrahydrocurcumin hadapproximately equal potencies in induction of phase II enzymes, ameasure of detoxification activity, whereas the fully saturatedtetrahydrocurcumin has little ability to induce phase II enzymes.Tetrahydrocurcumin, the curcumin derivative with the least antitumoractivity, caused a 1.6-fold induction of quinone reductase activity atthe highest concentration tested, 25 μM. However, TPCPD, which is anunsaturated aromatic ketone with no anti-angiogenic activity, had a CDvalue of 4.8 μM. The results are shown in Table 1. Thus, antiangiogenicactivity does not correlate with phase II activity. This finding isevidence that the two processes are not based on similar mechanisms.

Modifications and variations of the methods and compositions describedherein will be obvious to those skilled in the art and are intended tocome within the scope of the appended claims.

TABLE 1 Actual and Relative Effects of Curcuminoids and TPCPD On PhaseII Enzyme Induction and Angiogenisis ANTI-ANGIOGENIC EFFECT PHASE IISector Vessel INDUCTION Size³ Length COMPOUND CD¹ Rank² (μM) Rank² (mm.)Rank² Tetrahydro- >25 1 2.43 2 0.74 3 curcumin Bisdemethoxy- 11.0 2 1.73 0.88 2 curcumin Demethoxy- 9.0 3 0.71 5 0.26 5 curcumin Curcumin 7.3 41.17 4 0.59 4 TPCPD 4.8 5 3.72 1 1.16 1 Notes: ¹Concentration to doublethe measured specific activity; negatively correlated with effectiveness²Rank: Relative effectiveness in Phase II enzyme induction or inantiangiogenic effect (reduction of sector size or vessel length)³Sector size expresses in units of 1/12 of a circle, or 30 degrees(equivalent to “clock hours”)

1. A method for inhibiting disorders of the gastrointestinal tractcharacterized by elevated levels of basic fibroblast growth factorcomprising orally administering to an individual having agastrointestinal disorder an effective amount of a curcuminoid toinhibit the disorder. 2-7. (canceled)
 8. The method of claim 1 whereinthe curcuminoid is curcumin.
 9. The method of claim 1 wherein thecurcuminoid is selected from the group consisting of demethoxycurcumin,bisdemethoxycurcumin, and tetrahydrocurcumin. 10-16. (canceled)